The innate and adaptive immune systems are important in the pathogenesis of rheumatoid arthritis (RA). Toll- like receptors (TLRs) are important for the generation of adaptive immunity, including T and B cell activation. However the role of TLRs in the persistence and destruction of RA is not known. We recently demonstrated that mice expressing a mutant Fas receptor (lpr), developed less frequent and less severe collagen induced arthritis (CIA). We identified a novel mechanism whereby Fas-FasL interactions on macrophages (M&s), while not inducing apoptosis, enhanced activation through the TLR4 pathway. We demonstrated that intact Fas- FasL signaling promoted the induction of CIA and enhanced the NF-:B activation and cytokine expression induced by TLR4 ligation. M&s and synovial fibroblasts are critical in the pathogenesis of RA. M&s in the RA joint, express both Fas and FasL and are in intimate contact with other M&s and with synovial fibroblasts. These observations suggest that, in the RA joint, Fas-FasL interactions, by lowering the threshold for activation, may sensitize synovial M&s to activation by TLR ligands. Our preliminary studies also demonstrate that M&s isolated from the RA joint demonstrate increased responsiveness to microbial TLR2 and TLR4 ligands compared to control M&s, suggesting that TLR signaling might be important in RA. Our preliminary data also demonstrate that the 96-kDa glycoprotein 96 (gp96), an endoplasmic reticulum stress response protein, is highly expressed in the RA joint, and activates M&s through TLR2 and that non-apoptotic Fas-FasL interactions promote M& activation by gp96. We also demonstrate that another potential TLR ligand, HSP22, which activates through TLR4, is abundant in RA synovial fluids. Together these observations suggest that Fas-FasL interactions between M&s, and perhaps between M&s and synovial fibroblasts, may sensitize cells locally within the joint to activation by endogenous TLR ligands, such gp96 or HSP22, resulting in a self- perpetuating chronic inflammation. The overriding hypothesis of this application is that the local release of endogenous TLR ligands promotes the progression of arthritis from a self-limited to a chronic disease, and that this progression is enhanced by Fas-FasL interactions. Specifically, we hypothesize that: 1) Employing gp96 and HSP22, the activation of RA synovial M&s through TLR ligation will be greater than observed with normal M&s or those from the synovial fluid of patients with other forms of inflammatory arthritis (OIA) such as psoriatic arthritis, while the activation of RA synovial fibroblasts will be greater than observed with synovial fibroblasts from patients with osteoarthritis (OA); 2) that Fas-FasL interactions are required for maximal TLR2 or TLR4-mediated activation by endogenous TLR ligands; 3) that the expression of gp96 (or HSP22) converts self-limited arthritis to chronic progressive disease, dependent upon TLRs, in the presence, but not the absence, of intact Fas signaling and that 4) that neutralization of gp96 (and/or HSP22) will ameliorate CIA. PUBLIC HEALTH RELEVANCE. Rheumatoid Arthritis affects at least 1% of the population and there are many patients for whom therapy is inadequate. The work proposed in this study is aimed at identifying novel mechanisms contributing to the pathogenesis of rheumatoid arthritis, which would define new therapeutic targets.